ATM is a member of the PI(3)K (phosphatidyl-inositol-3-OH kinase)-like kinase (PIKKs) family, that also includes the DNA dependent protein kinase (DNA-PK). DNA-PK is a core component of the non-homologous end joining pathway that is required to join DNA double strand breaks together. This project is based on our finding that the activity of DNA-PK partially substitutes for the function of ATM (E. Callen, 2008 submitted). For example, we have found that ATM and DNA-PK carry out redundant roles in DNA repair and activation of the tumor suppressor p53. Nevertheless, despite the fact that ATM and DNA-PK are both stimulated by the same lesions (a double strand break), recruited with similar kinetics, and have similar substrates specificity, in vivo phosphorylations are more severely compromised by loss of ATM. We hypothesize that there might be compounds that INCREASE that activity of DNA-PK or its downstream pathway (or interfere with inhibitors of the pathway), which in turn would promote DNA repair. Recent studies indicate that DNA breaks can be also channeled into alternative DNA-end joining pathways, (A. Nussenzweig et al. Cell 2007, 131:223) which might be targeted by small-molecule compounds. To assay for compounds that promote DNA repair, we will use a robust experimental approach that tracks the repair of physiological DNA breaks generated during V(D)J recombination. This process is initiated by the recombinase activating gene RAG endonuclease. Abelson kinase-transformed pre-B-cell lines (ATM+/+, ATM-/-) have been generated in Barry Sleckmans laboratory (Bredemeyer, A et al Nature 442, 466-470 2006), who is a collaborator on this project. Treatment of pre-B-cell lines with the Abelson kinase inhibitor, STI571 (ie. Gleevac), leads to a rapid induction of RAG gene expression. Pre-B-cell lines have been transduced with the retroviral recombination reporter pMX-INV. This reporter substrate has a single pair of recombination signals targeted by RAG that flank an anti-sense green fluorescent protein (GFP) cDNA and mediate recombination by inversion. Thus, normal rearrangement mediated by non-homologous end joining places the GFP cDNA in the sense orientation, allowing for GFP expression. Analysis of pMX-INV rearrangement in STI571-treated Atm-/-:INV cells